Lysophosphatidic acid does not cause blood/lymphatic vessel plasticity in the rat mesentery culture model.

Understanding the mechanisms behind endothelial cell identity is crucial for the goal of manipulating microvascular networks. Lysophosphatidic acid (LPA) and serum stimulation have been suggested to induce a lymphatic identity in blood endothelial cells in vitro. The objective of this study was to determine if LPA or serum induces blood-to-lymphatic vessel phenotypic transition in microvascular networks. The rat mesentery culture model was used to observe the effect of stimulation on blood and lymphatic microvascular networks ex vivo. Vascularized mesenteric tissues were harvested from adult Wistar rats and cultured with LPA or 10% serum for up to 5 days. Tissues were then immunolabeled with PECAM to identify blood vessels and LYVE-1 or Prox1 to identify lymphatic vessels. We show that while LPA caused capillary sprouting and increased vascular length density in adult microvascular networks, LPA did not cause a blood-to-lymphatic phenotypic transition. The results suggest that LPA is not sufficient to cause blood endothelial cells to adopt a lymphatic identity in adult microvascular networks. Similarly, serum stimulation caused robust angiogenesis and increased lymphatic/blood vessel connections, yet did not induce a blood-to-lymphatic phenotypic transition. Our study highlights an understudied area of lymphatic research and warrants future investigation into the mechanisms responsible for the maintenance of blood and lymphatic vessel identity.

Printing cancer cells into intact microvascular networks: a model for investigating cancer cell dynamics during angiogenesis.

While cancer cell invasion and metastasis are dependent on cancer cell-stroma, cancer cell-blood vessel, and cancer cell-lymphatic vessel interactions, our understanding of these interactions remain largely unknown. A need exists for physiologically-relevant models that more closely mimic the complexity of cancer cell dynamics in a real tissue environment. The objective of this study was to combine laser-based cell printing and tissue culture methods to create a novel ex vivo model in which cancer cell dynamics can be tracked during angiogenesis in an intact microvascular network. Laser direct-write (LDW) was utilized to reproducibly deposit breast cancer cells (MDA-MB-231 and MCF-7) and fibroblasts into spatially-defined patterns on cultured rat mesenteric tissues. In addition, heterogeneous patterns containing co-printed MDA-MB-231/fibroblasts or MDA-MB-231/MCF-7 cells were generated for fibroblast-directed and collective cell invasion models. Printed cells remained viable and the cells retained the ability to proliferate in serum-rich media conditions. Over a culture period of five days, time-lapse imaging confirmed fibroblast and MDA-MB-231 cell migration within the microvascular networks. Confocal microscopy indicated that printed MDA-MB-231 cells infiltrated the tissue thickness and were capable of interacting with endothelial cells. Angiogenic network growth in tissue areas containing printed cancer cells was characterized by significantly increased capillary sprouting compared to control tissue areas containing no printed cells. Our results establish an innovative ex vivo experimental platform that enables time-lapse evaluation of cancer cell dynamics during angiogenesis within a real microvascular network scenario.

Pericyte dynamics during angiogenesis: new insights from new identities.

Therapies aimed at manipulating the microcirculation require the ability to control angiogenesis, defined as the sprouting of new capillaries from existing vessels. Blocking angiogenesis would be beneficial in many pathologies (e.g. cancer, retinopathies and rheumatoid arthritis). In others (e.g. myocardial infarction, stroke and hypertension), promoting angiogenesis would be desirable. We know that vascular pericytes elongate around endothelial cells (ECs) and are functionally associated with regulating vessel stabilization, vessel diameter and EC proliferation. During angiogenesis, bidirectional pericyte-EC signaling is critical for capillary sprout formation. Observations of pericytes leading capillary sprouts also implicate their role in EC guidance. As such, pericytes have recently emerged as a therapeutic target to promote or inhibit angiogenesis. Advancing our basic understanding of pericytes and developing pericyte-related therapies are challenged, like in many other fields, by questions regarding cell identity. This review article discusses what we know about pericyte phenotypes and the opportunity to advance our understanding by defining the specific pericyte cell populations involved in capillary sprouting.

VEGF-C induces lymphangiogenesis and angiogenesis in the rat mesentery culture model.

OBJECTIVE: Lymphatic and blood microvascular systems are critical for tissue function. Insights into the coordination of both systems can be gained by investigating the relationships between lymphangiogenesis and angiogenesis. Recently, our laboratory established the rat mesentery culture model as a novel tool to investigate multicellular interactions during angiogenesis in an intact microvascular network scenario. The objective of this study was to determine whether the rat mesentery culture model can be used to study lymphangiogenesis. METHODS: Mesenteric tissue windows were harvested from adult male Wistar rats and cultured for three or five days in either serum-free MEM or MEM supplemented with VEGF-C. Tissues were immunolabeled for PECAM and LYVE-1 to identify blood and lymphatic endothelial cells, respectively. Tissues selected randomly from those containing vascular networks were quantified for angiogenesis and lymphangiogenesis. RESULTS: VEGF-C treatment resulted in an increase in the density of blood vessel sprouting compared to controls by day 3. By day 5, lymphatic sprouting was increased compared to controls. CONCLUSIONS: These results are consistent with in vivo findings that lymphangiogenesis lags angiogenesis after chronic stimulation and establish a tool for investigating the interrelationships between lymphangiogenesis and angiogenesis in a multisystem microvascular environment.

Relationships between lymphangiogenesis and angiogenesis during inflammation in rat mesentery microvascular networks.

BACKGROUND: Lymphatic and blood microvascular systems play a coordinated role in the regulation of interstitial fluid balance and immune cell trafficking during inflammation. The objective of this study was to characterize the temporal and spatial relationships between lymphatic and blood vessel growth in the adult rat mesentery following an inflammatory stimulus. METHODS AND RESULTS: Mesenteric tissues were harvested from unstimulated adult male Wistar rats and at 3, 10, and 30 days post compound 48/80 stimulation. Tissues were immunolabeled for PECAM, LYVE-1, Prox1, podoplanin, CD11b, and class III ß-tubulin. Vascular area, capillary blind end density, and vascular length density were quantified for each vessel system per time point. Blood vascular area increased compared to unstimulated tissues by day 10 and remained increased at day 30. Following the peak in blood capillary sprouting at day 3, blood vascular area and density increased at day 10. The number of blind-ended lymphatic vessels and lymphatic density did not significantly increase until day 10, and lymphatic vascular area was not increased compared to the unstimulated level until day 30. Lymphangiogenesis correlated with the upregulation of class III ß-tubulin expression by endothelial cells along lymphatic blind-ended vessels and increased lymphatic/blood endothelial cell connections. In local tissue regions containing both blood and lymphatic vessels, the presence of lymphatics attenuated blood capillary sprouting. CONCLUSIONS: Our work suggests that lymphangiogenesis lags angiogenesis during inflammation and motivates the need for future investigations aimed at understanding lymphatic/blood endothelial cell interactions. The results also indicate that lymphatic endothelial cells undergo phenotypic changes during lymphangiogenesis.